scholarly journals Hypoxanthine Guanine Phosphoribosyl Transferase Is the Most Stable Reference Gene for Gene Expression Analysis by Quantitative PCR in Peripheral Blood Mononuclear Cells from Women with the Polycystic Ovary Syndrome

2014 ◽  
Vol 33 (4) ◽  
pp. 356-363 ◽  
Author(s):  
Danijela Vojnović Milutinović ◽  
Djuro Macut ◽  
Ivana Božić Antić ◽  
Jelica Bjekić Macut ◽  
Marina Nikolić ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Mononuclear Cells ◽  
Polycystic Ovary ◽  
Normal Weight ◽  
Expression Stability ◽  
Rt Pcr ◽  
Ovary Syndrome ◽  
Peripheral Blood Mononuclear ◽  
Software Packages

Summary Background: The polycystic ovary syndrome (PCOS) is a frequent endocrine disorder that affects women of reproductive age. As the syndrome is strongly associated with obesity, it is of interest to examine the gene expression diffe rences that accompany its development and the associ a ted metabolic disturbances. Real-time RT PCR is a standard method for studying changes in gene expression. However, to obtain accurate and reliable results, validation of reference genes is obligatory. The aim of this study was to identify a suitable reference for the normalization of gene expression in peripheral blood mononuclear cells (PBMCs) from obese and normal-weight women with PCOS. Methods: The expression stability of four potential reference genes: hypoxanthine guanine phosphoribosyl trans-ferase 1 (HPRT), β-actin (BA), β2-microglobulin (B2M) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was assessed in PBMCs from healthy women, and from normal-weight and obese women with PCOS. The variability in the expression of potential reference genes was analyzed by the TaqMan real-time RT PCR method, using GeNorm and NormFinder software packages. Results: Direct comparison of cycle threshold (Ct) values showed inter-individual variations for all validated genes, the Ct values of HPRT being less variable than those of BA, GAPDH and B2M. Both software packages pointed to HPRT as the most steadily expressed gene in the PBMCs of women with PCOS and healthy controls. Conclusions: Cross-validation of the expression stability of four potential reference genes identified HPRT as the most stable reference, suitable for further investigations of gene expression in PBMCs from women with PCOS.

scholarly journals Quantitative Evaluation and Selection of Reference Genes for Quantitative RT-PCR in Mouse Acute Pancreatitis

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Zhaoping Yan ◽  
Jinhang Gao ◽  
Xiuhe Lv ◽  
Wenjuan Yang ◽  
Shilei Wen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Pancreatitis ◽  
Reference Gene ◽  
Reference Genes ◽  
Suitable Reference Gene ◽  
Expression Stability ◽  
Rt Pcr ◽  
Comprehensive Method ◽  
Suitable Combination ◽  
Suitable Reference

The analysis of differences in gene expression is dependent on normalization using reference genes. However, the expression of many of these reference genes, as evaluated by quantitative RT-PCR, is upregulated in acute pancreatitis, so they cannot be used as the standard for gene expression in this condition. For this reason, we sought to identify a stable reference gene, or a suitable combination, for expression analysis in acute pancreatitis. The expression stability of 10 reference genes (ACTB, GAPDH, 18sRNA, TUBB, B2M, HPRT1, UBC, YWHAZ, EF-1α, and RPL-13A) was analyzed using geNorm, NormFinder, and BestKeeper software and evaluated according to variations in the raw Ct values. These reference genes were evaluated using a comprehensive method, which ranked the expression stability of these genes as follows (from most stable to least stable): RPL-13A, YWHAZ > HPRT1 > GAPDH > UBC > EF-1α> 18sRNA > B2M > TUBB > ACTB. RPL-13A was the most suitable reference gene, and the combination of RPL-13A and YWHAZ was the most stable group of reference genes in our experiments. The expression levels of ACTB, TUBB, and B2M were found to be significantly upregulated during acute pancreatitis, whereas the expression level of 18sRNA was downregulated. Thus, we recommend the use of RPL-13A or a combination of RPL-13A and YWHAZ for normalization in qRT-PCR analyses of gene expression in mouse models of acute pancreatitis.

Plasmatic and Intracellular Markers of Oxidative Stress in Normal Weight and Obese Patients with Polycystic Ovary Syndrome

2017 ◽  
Vol 125 (08) ◽  
pp. 506-513 ◽  
Author(s):  
Chantal Di Segni ◽  
Andrea Silvestrini ◽  
Romana Fato ◽  
Christian Bergamini ◽  
Francesco Guidi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Insulin Resistance ◽  
Polycystic Ovary Syndrome ◽  
Mononuclear Cells ◽  
Polycystic Ovary ◽  
Gradient Centrifugation ◽  
Normal Weight ◽  
Model Assessment ◽  
Obese Patients ◽  
Ovary Syndrome

Abstract Introduction Insulin resistance (IR) is associated with polycystic ovary syndrome (PCOS). Oxidative stress (OS) is, in turn, related to IR. Studies in PCOS evidenced an increase in OS markers, but they are mainly performed in obese patients, while the complex picture of normal weight PCOS is still poorly investigated. Matherials and Methods To investigate OS in PCOS and relationship with hormonal and metabolic picture, we performed a case-control study in 2 PCOS groups: normal weight (N-PCOS, n=21, age 18–25 ys, mean±SEM BMI 20.7±0.2 kg/m2) and obese (OB-PCOS, n=15, 20–30 ys, BMI 32.8±1.1), compared with control groups matched for BMI: normal (N-C, n=10, 20–30 ys, BMI 21.6±0.9) and obese (OB-C, n=20, 21–31ys, BMI 36.8±1.0). Malondialdehyde (MDA) in blood plasma and peripheral mononuclear cells, obtained by density-gradient centrifugation, was assayed spectrophotometrically by TBARS assay. CoenzymeQ10 (CoQ10) in plasma and cells was assayed by HPLC. Plasma Total Antioxidant Capacity (TAC) was also measured by spectrophotometric method. Results PCOS patients exhibited higher Testosterone levels than controls, but OB-PCOS had highest HOMA (Homeostasis Model Assessment) index, suggesting marked insulin resistance. Despite plasma MDA levels were not significantly different (N-PCOS 3380±346.94 vs. N-C 7 120±541.66; OB-PCOS 5 517.5±853.9 vs. OB. 3 939.66±311.2 pmol/ml), intracellular MDA levels were significantly higher in N-PCOS than controls (mean 3 259±821.5 vs. 458±43.2 pmol/106/cells) and higher than OB-PCOS, although not significantly (1363.1±412.8 pmol/106/cells). Intracellular CoenzymeQ10 was higher in N-PCOS than in N-C, but the highest levels were found in OB-C. Conclusions Our data, while confirming the presence of OS in obese PCOS patients in agreement with literature, suggest that OS could be present also in normal weight PCOS, but it can be revealed in tissue rather than in plasma. The relationship with metabolic status remains to be established, but could be a physiopathological basis for antioxidant treatment in such patients.

scholarly journals OR20-03 Transcriptional Changes in Lipid Metabolism of Adipocytes Derived from Subcutaneous Abdominal Adipose Stem Cells of Normal-Weight Polycystic Ovary Syndrome Women

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Karen Lynn Leung ◽  
Smriti Sanchita ◽  
Phillip Dumesic ◽  
Xiangming Ding ◽  
Xinmin Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Polycystic Ovary Syndrome ◽  
Polycystic Ovary ◽  
Normal Weight ◽  
Beta Oxidation ◽  
Ovary Syndrome ◽  
Time Points ◽  
Upstream Regulator ◽  
Regulator Genes

Abstract Normal-weight polycystic ovary syndrome (PCOS) women exhibit increased adipose insulin resistance in vivo (1) accompanying enhanced subcutaneous (SC) abdominal adipose stem cell (ASC) development to adipocytes with greater lipid accumulation per cell in vitro (2). To determine whether this phenomenon is associated with abnormal adipogenic gene transcription during ASC differentiation into adipocytes, SC abdominal ASCs isolated from three non-Hispanic Caucasian normal-weight PCOS women and three age- and BMI-matched controls were cultured in adipogenic differentiation medium for 3–12 days. After RNA isolation, gene expression levels were determined by RNA sequencing at days 3, 7, and 12. Differentially expressed genes were filtered for significance (padj<0.05) and fold change (>2-fold); upstream regulator genes and gene ontology (GO) functions were determined using Ingenuity Pathway Analysis. Gene set enrichment analysis (GSEA) also was used to identify enriched cellular processes (3). Differentially expressed genes in PCOS vs. control cells were either upregulated (466, 768 and 441 genes on days 3, 7 and 12, respectively) or downregulated (742, 974 and 605 genes on days 3, 7 and 12, respectively) over time, with critical genes governing adipocyte cell differentiation in PCOS cells increased 2–6 fold at days 3, 7 and 12 (PPARγ, CEBPα, ADIPOQ, AGPAT2, FABP4, LPL, PLIN1). The predicted upstream regulator genes TGFβ1 (an adipogenic inhibitor) and TNF (a pro-inflammatory adipokine) were significantly reduced in PCOS relative to control cells at all time points. The GO functions lipid oxidation and free fatty acid (FFA) beta-oxidation were enriched amongst upregulated genes in PCOS cells across all time points, while acylglycerol synthesis was increased at days 7 and 12 alone (z>2, p<0.05, all GO functions). In parallel, GSEA showed in PCOS cells significantly increased transcripts related to oxidative phosphorylation, peroxisome activity and adipogenesis at all time points (p<0.05). Thus, adipocytes derived from SC abdominal ASCs of normal-weight PCOS women exhibit early activation of adipogenic genes, potentially underlying their exaggerated lipid accumulation in vitro, as previously described (2). These PCOS-related changes in gene expression involve an increase in both oxidative phosphorylation and FFA beta oxidation, which could disrupt the balance between energy production and lipid storage, particularly when caloric intake exceeds energy utilization. References: (1) Dumesic DA, et al JCEM 2019;104(6):2171–83; (2) Leung KL, et al. JES 2019;3:Supplement 1, SUN-107 (3) Subhramanian A, et al. PNAS 2005;102:43

scholarly journals Characteristics of a Novel Target Antigen against Myeloma Cells for Immunotherapy

2020 ◽  
Author(s):  
Maiko Matsushita ◽  
Saku Saito ◽  
Shinya Yokoe ◽  
Daiju Ichikawa ◽  
Yutaka Hattori
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Target Antigen ◽  
Rt Pcr ◽  
Peripheral Blood Mononuclear ◽  
Myeloma Cells ◽  
Demethylating Agents ◽  
Specific Ctls

Despite the availability of therapeutic treatments, multiple myeloma is an incurable haematological disorder. In this study, we aimed to clarify the role of CXorf48 as a therapeutic target in multiple myeloma. Based on a previously identified HLA-A*24:02-restiricted epitope from this novel cancer/testis antigen, we characterized the activities of cytotoxic T lymphocytes (CTLs) specific to this antigen against myeloma cells and evaluated the effects of demethylating agents in increasing antigen expression and enhancing the cytotoxic activity of CTLs. CXorf48 expression was examined by RT-PCR using nine myeloma cell lines. Cell lines with low CXorf48 expression were treated by demethylating agents (DMAs), 5-azacytidine (5-aza), and 5-aza-2'-deoxycytidine (DAC) to evaluate gene expression using quantitative RT-PCR. Furthermore, CXorf48-specific CTLs were induced from peripheral blood mononuclear cells of HLA-A*24:02-positive healthy donors to evaluate antigen recognition using ELISpot and 51Cr cytotoxicity assays. CXorf48 was widely expressed in myeloma cells and gene expression was significantly increased by DMAs. Furthermore, CXorf48-specific CTLs recognized DMA-treated myeloma cells. These findings suggest that CXorf48 is a useful target for immunotherapy, such as vaccination, in combination with demethylating agents for the treatment of patients with myeloma.

scholarly journals Characteristics of a Novel Target Antigen against Myeloma Cells for Immunotherapy

Vaccines ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 579
Author(s):  
Maiko Matsushita ◽  
Saku Saito ◽  
Shinya Yokoe ◽  
Daiju Ichikawa ◽  
Yutaka Hattori
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Target Antigen ◽  
Rt Pcr ◽  
Peripheral Blood Mononuclear ◽  
Myeloma Cells ◽  
Demethylating Agents ◽  
Specific Ctls

Despite the availability of therapeutic treatments, multiple myeloma is an incurable haematological disorder. In this study, we aimed to clarify the role of CXorf48 as a therapeutic target in multiple myeloma. Based on a previously identified HLA-A*24:02-restiricted epitope from this novel cancer/testis antigen, we characterized the activities of cytotoxic T lymphocytes (CTLs) specific to this antigen against myeloma cells and evaluated the effects of demethylating agents in increasing antigen expression and enhancing the cytotoxic activity of CTLs. CXorf48 expression was examined by reverse transcription polymerase chain reaction (RT-PCR) using nine myeloma cell lines. Cell lines with low CXorf48 expression were treated by demethylating agents (DMAs), 5-azacytidine (5-aza), and 5-aza-2’-deoxycytidine (DAC) to evaluate gene expression using quantitative RT-PCR. Furthermore, CXorf48-specific CTLs were induced from peripheral blood mononuclear cells of HLA-A*24:02-positive healthy donors to evaluate antigen recognition using ELISpot and 51Cr cytotoxicity assays. CXorf48 was widely expressed in myeloma cells, and gene expression was significantly increased by DMAs. Furthermore, CXorf48-specific CTLs recognized DMA-treated myeloma cells. These findings suggest that CXorf48 is a useful target for immunotherapy, such as vaccination, in combination with demethylating agents for the treatment of patients with myeloma.

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